1) Field of the Invention
The invention described herein relates to the field of three-dimensional cell culture scaffolds.
2) Description of Related Art
Three dimensional (3D) cultures provide innovative approaches to study processes that contribute to tumorigenesis because they recapitulate cancer cells in their native in vivo environment. The majority of the supporting data that posits the importance of tumorigenesis has been obtained using two dimensional (2D) cell culture systems. Cells in 2D are subjected to unnatural mechanical and geometric constraints that do not represent the three dimensional (3D) milieu of a tumor. The complex interplay between biochemical, and mechanical properties may be undermined or compromised in 2D cultures and may affect many important functions such as gene and protein expression. Considerations on the mechanical, biochemical and physical properties of any 3D system, aim to mimic the native ECM. One of the major advantages is the potential for rapid experimental manipulation achieved by controlling these parameters that can permit development of sophisticated cancer models. Tailored 3D cell culture scaffolds combining relevant platforms with multiple bio-functionalities will allow for the specific induction of signal transduction pathways, the sorting of different cell types, or the control of cancer cell differentiation. Combination of existing 3D systems that impart separate yet important characteristics that preserve structural and functional in vivo-like complexity to the whole will increase the sophistication of these 3D models. Presently, there exist a significant need for more realistic tumor models to study tumorigenesis and the effective screening of anticancer drugs.
The most commonly used 3D model are spheroids that are used for a variety of experimental studies in chemotherapy and radiotherapy and are being pursued in high throughput screening (HTS) programs for drug development, candidate efficacy and safety. Spheroids impart functional and mass transport properties similar to those observed in micrometastases or poorly vascularized regions in solid tumors. [Hirschhaeuser F, et al., Multicellular tumor spheroids: An underestimated tool is catching up again. Journal of biotechnology. 2010; 148:3-15.] These features combined with the complexities of cell-cell and cell-matrix interactions, affect the uptake, penetration, distribution and bioactivity of therapeutic drugs. They are simple 3D structures that can be generated from a wide range of cell types, and form due to the tendency of adherent cells to aggregate and are typically created from single or co-culture techniques such as hanging drop, rotating culture or conclave plate methods to name a few. [Pampaloni F, et al., The third dimension bridges the gap between cell culture and live tissue. Nature Reviews Molecular Cell Biology. 2007; 8:839-45; Timmins N E, et al., Method for the generation and cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix. Cell and tissue research. 2005; 320:207-10; Castaneda F, and Kinne R K H. Short exposure to millimolar concentrations of ethanol induces apoptotic cell death in multicellular HepG2 spheroids. Journal of cancer research and clinical oncology. 2000; 126:305-10.]
The inherent limitation of this model is that it is entirely cell based and do not represent the mechanical features of the ECM as a whole. To address this issue various substrates or scaffolds derived from biological, natural or synthetic sources have been used to form hydrogels, films, fibers, micromolded structures in microfluidic devices, and microchips in the construction of spheroids. For example, hepatocytes self-assemble to form spheroids on scaffolds made from alginate, hyaluronic acid, peptide scaffolds, and galactosylated meshes. [Gurski L A, et al., Hyaluronic acid-based hydrogels as 3D matrices for in vitro evaluation of chemotherapeutic drugs using poorly adherent prostate cancer cells (vol 30, pg 6076, 2009). Biomaterials. 2010; 31:4248; Elkayam T, et al., Enhancing the drug metabolism activities of C3A-A human hepatocyte cell line—By tissue engineering within alginate scaffolds. Tissue engineering. 2006; 12:1357-68; Shin J Y, et al. Efficient formation of cell spheroids using polymer nanofibers. Biotechnology letters. 2012; 34:795-803; Wang D D, et al. Thermoreversible Hydrogel for In Situ Generation and Release of HepG2 Spheroids. Biomacromolecules. 2011; 12:578-84; Chung T W, et al. Preparation of alginate/galactosylated chitosan scaffold for hepatocyte attachment. Biomaterials. 2002; 23:2827-34; Ivascu A, and Kubbies M. Diversity of cell-mediated adhesions in breast cancer spheroids. International journal of oncology. 2007; 31:1403-13; Horning J L, et al. 3-D tumor model for in vitro evaluation of anticancer drugs. Molecular pharmaceutics. 2008; 5:849-62; Semino C E, et al., Functional differentiation of hepatocyte-like spheroid structures from putative liver progenitor cells in three-dimensional peptide scaffolds. Differentiation; research in biological diversity. 2003; 71:262-70; Chua K N, et al., Stable immobilization of rat hepatocyte spheroids on galactosylated nanofiber scaffold. Biomaterials. 2005; 26:2537-47.]
Incorporation of spheroids into synthetic 3D polymeric scaffolds has been used as a model for screening anticancer drugs. [Ho W J, et al. Incorporation of multicellular spheroids into 3-D polymeric scaffolds provides an improved tumor model for screening anticancer drugs. Cancer science. 2010; 101:2637-43.] These scaffolds provide support for the spheroids thereby mimicking the physical interaction of the tumor with the topographical features of the native ECM, as for example, between the tumor and the basement membrane.
The interaction of mammalian cells with sub cellular topography has proven to be an important signaling modality in controlling cell function via mechanotransductive cues. The tumor microenvironment consisting of tumor cells and corresponding stroma intimately associate with the physical structures of the ECM during all stages of tumorigenesis. Synthetic substrate topography has been shown to influence cell migration, differentiation, and gene expression. For example, SAL/N cancer fibroblasts cultured on micropatterned PDMS and C6 glioma cells cultured on polystyrene periodic structures exhibit differences in morphology, proliferation and migration in response to various topographical cues. [Tzvetkova-Chevolleau T, et al., The motility of normal and cancer cells in response to the combined influence of the substrate rigidity and anisotropic microstructure. Biomaterials. 2008; 29:1541-51; Wang X F, et al., Influence of physicochemical properties of laser-modified polystyrene on bovine serum albumin adsorption and rat C6 glioma cell behavior. Journal of Biomedical Materials Research Part A. 2006; 78A:746-54.]
Electrospinning is a versatile technique used to produce polymeric fibrous scaffolds for cell culture applications. It allows for the preparation of unique matrices of aligned or non-woven meshes containing nano to micrometer sized fibers using diverse materials and fabrication techniques. [Zanatta G, et al., Viability of mesenchymal stem cells during electrospinning Brazilian journal of medical and biological research=Revista brasileira de pesquisas medicas e biologicas/Sociedade Brasileira de Biofisica [et al]. 2012; 45:125-30; Zanatta G, et al., Mesenchymal stem cell adherence on poly(D, L-lactide-co-glycolide) nanofibers scaffold is integrin-beta 1 receptor dependent. Journal of biomedical nanotechnology. 2012; 8:211-8; Yu D G, et al., Modified coaxial electrospinning for the preparation of high-quality ketoprofen-loaded cellulose acetate nanofibers. Carbohydrate polymers. 2012; 90:1016-23; Tsai S W, et al. MG63 osteoblast-like cells exhibit different behavior when grown on electrospun collagen matrix versus electrospun gelatin matrix. PloS one. 2012; 7:e31200; Sundaramurthi D, et al., Electrospun nanostructured chitosan-poly(vinyl alcohol) scaffolds: a biomimetic extracellular matrix as dermal substitute. Biomedical materials. 2012; 7:045005; Samavedi S, et al.; Response of bone marrow stromal cells to graded co-electrospun scaffolds and its implications for engineering the ligament-bone interface. Biomaterials. 2012; Meinel A J, et al., Electrospun matrices for localized drug delivery: current technologies and selected biomedical applications. European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik eV. 2012; 81:1-13.] Studies have shown that modified electrospun scaffolds simulate favorable functional responses in cancer cells [Agudelo-Garcia P A, et al., Glioma Cell Migration on Three-dimensional Nanofiber Scaffolds Is Regulated by Substrate Topography and Abolished by Inhibition of STAT3 Signaling. Neoplasia. 2011; 13:831-U96; Johnson J, et al., Quantitative Analysis of Complex Glioma Cell Migration on Electrospun Polycaprolactone Using Time-Lapse Microscopy. Tissue Engineering Part C-Methods. 2009; 15:531-40; Xie J W, and Wang C H. Electrospun micro- and nanofibers for sustained delivery of paclitaxel to treat C6 glioma in vitro. Pharmaceutical research. 2006; 23:1817-26.]